Method for highly sensitive detection of protein-protein interaction

ABSTRACT

The present invention intends to provide an assay system using split luciferase that has a remarkably high detection sensitivity. In an embodiment, binding of mutually binding first and second proteins is detected by preparing a first fusion protein comprising the first protein fused with a peptide having the amino acid sequence of amino acid SEQ ID NO: 1 and a second fusion protein comprising the second protein fused with a peptide having an amino acid sequence selected from the group consisting of amino acid SEQ ID NOS: 2 to 6, and allowing the first fusion protein to bind with the second fusion protein to form a complex, and detecting luminescence emitted from the complex.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage entry under 35 USC §371(b) ofPCT International Application Serial No. PCT/JP2010/059160, filed May28, 2010, which claims priority to Japanese Patent Application SerialNumber 2009-131481, filed May 29, 2009 and Japanese Patent ApplicationSerial Number 2010-037921, filed Feb. 23, 2010, the disclosures of allof which are hereby incorporated herein by reference.

TECHNICAL FIELD

This invention relates to methods for detecting a protein-proteininteraction.

BACKGROUND ART

A method for detecting protein interaction between two target proteinsby using complementarity of split luciferase fragments has been recentlydeveloped (Kim, S. B., Ozawa, T., Watanabe, S., Umezawa, Y., 2004. Proc.Natl. Acad. Sci. USA. 101, 11542-11547). The method for detecting aprotein-protein interaction using complementarity is generally conductedby fusing fragments of a split reporter protein respectively with thetarget proteins, and in this process, each fragment does not havesignificant activity by itself. When the target proteins interact witheach other, the inactive reporter protein fragments complement with eachother to regain the activity which allows emission of the signal toenable indirect tracking of the protein-protein interaction.

Such method using the complementarity has been used for various reporterproteins such as dihydrofolate reductase and β-lactamase greenfluorescent protein. Also, several luciferases such as Renillareniformis luciferase, Photinus Pyralis luciferase, red-emittingPhotinus Pyralis luciferase, and green-emitting Photinus Pyralisluciferase have been used.

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide an assay system usingsplit luciferase which has remarkably high detection sensitivity.

Solution to Problem

The inventors of the present invention made an intensive study forsolving the problem as described above, and found that, when usingluciferase from Brazilian larval click-beetle (Pyrearinustermitilluminans), a C terminal fragment having SEQ ID NO: 1 and an Nterminal fragment having any one of SEQ ID NOS: 2 to 6 are fused witheach of the two interacting proteins respectively, and the two fusionproteins are bound, a luminescence with an intensity about 30 foldstronger than the conventional assay is emitted. The present inventionhas been completed on the bases of such a finding.

Accordingly, one aspect of the present invention is a fusion proteinhaving the amino acid sequence of amino acid SEQ ID NO: 1. Anotheraspect of the present invention is a fusion protein having an amino acidsequence selected from the group consisting of amino acid SEQ ID NOS: 2to 6.

In the present specification, the “protein has an amino acid sequence”means that the protein contains the amino acid sequence and that theprotein may contain an amino acid sequence other than such an amino acidsequence. The “fusion protein” means a peptide derived from Pyrearinustermitilluminans luciferase (which, in the present invention, is apeptide consisting of an amino acid sequence selected from the groupconsisting of amino acid SEQ ID NO: 1 to 6) fused with a peptide notderived from Pyrearinus termitilluminans luciferase.

A further aspect of the invention is a complex of a fusion proteinhaving the amino acid sequence of amino acid SEQ ID NO: 1 and a fusionprotein having an amino acid sequence selected from the group consistingof amino acid SEQ ID NOS: 2 to 6.

A still further aspect of the invention is a DNA coding for an aminoacid sequence selected from the group consisting of amino acid SEQ IDNOS: 1 to 6, or may be an expression vector that contains this DNA andis capable of expressing a fusion protein having an amino acid sequenceselected from the group consisting of amino acid SEQ ID NOS: 1 to 6.

A still further aspect of the invention is a kit for detectingprotein-protein interaction containing an expression vector forexpressing a protein having a peptide comprising the amino acid sequenceof amino acid SEQ ID NO: 1 and an expression vector for expressing aprotein having a peptide comprising an amino acid sequence selected fromthe group consisting of amino acid SEQ ID NOS: 2 to 6.

A still further aspect of the invention is a method for detecting afusion protein having the amino acid sequence of amino acid SEQ ID NO:1, comprising the steps of allowing the fusion protein to interact witha binding fusion protein wherein the binding fusion protein has an aminoacid sequence selected from the group consisting of amino acid SEQ IDNOS: 2 to 6 and is capable of binding with the fusion protein, to allowformation of a complex, and detecting luminescence emitted from thecomplex.

A still further aspect of the invention is a method for detecting afusion protein containing an amino acid sequence selected from the groupconsisting of amino acid SEQ ID NOS: 2 to 6, comprising the steps ofallowing the fusion protein to interact with a binding fusion proteinwherein the binding fusion protein has the amino acid sequence of aminoacid SEQ ID NO: 1 and is capable of binding with the fusion protein, toform a complex, and detecting luminescence emitted from the complex.

A still further aspect of the invention is a method for detecting acomplex of a first fusion protein and a binding fusion protein beingcapable of binding with the first fusion protein, comprising the step ofdetecting luminescence emitted from the complex, wherein the firstfusion protein has the amino acid sequence of amino acid SEQ ID NO: 1and the binding fusion protein has an amino acid sequence selected fromthe group consisting of amino acid SEQ ID NOS: 2 to 6.

A still further aspect of the invention is a method for detectingbinding of first and second fusion proteins which are bound to eachother, wherein the first fusion protein has the amino acid sequence ofamino acid SEQ ID NO: 1 and the second fusion protein has an amino acidsequence selected from the group consisting of amino acid SEQ ID NOS: 2to 6, comprising the steps of allowing the first fusion protein tointeract with the second fusion protein to allow formation of a complex,and detecting luminescence emitted from the complex. This method mayfurther comprise the steps of fusing the amino acid sequence of aminoacid SEQ ID NO: 1 with a first protein to prepare the first fusionprotein, and fusing an amino acid sequence selected from the groupconsisting of amino acid SEQ ID NOS: 2 to 6 with a second protein toprepare the second fusion protein.

A still further aspect of the invention is a method for screening afusion protein library for a binding fusion protein being capable ofbinding to a first fusion protein, comprising the steps of allowing thefirst fusion protein to interact with a plurality of second fusionproteins, wherein the first fusion protein has the amino acid sequenceof amino acid SEQ ID NO: 1 and the second fusion proteins have an aminoacid sequence selected from the group consisting of amino acid SEQ IDNOS: 2 to 6 and are in the fusion protein library, and identifying thebinding fusion protein that forms a complex with the first fusionprotein by detecting luminescence emitted by the complex.

A still further aspect of the invention is a method for screening for abinding fusion protein being capable of binding with a first fusionprotein comprising the steps of allowing the first fusion protein tointeract with a plurality of second fusion proteins, wherein the firstfusion protein has an amino acid sequence selected from the groupconsisting of amino acid SEQ ID NOS: 2 to 6, and the second fusionproteins have the amino acid sequence of amino acid SEQ ID NO: 1, andidentifying the binding fusion protein that forms a complex with thefirst fusion protein detecting luminescence emitted from the complex.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is the nucleotide sequence of the cDNA of Pyrearinustermitilluminans luciferase.

FIG. 1B is a list of PCR primers used in preparing plucN and plucC inone example of the present invention.

FIG. 1C is the nucleotide sequence of the DNA inserted in multicloningsite of pcDNA3.1 in pcDNA3.1/myc-His(B).

FIG. 1D is the nucleotide sequence of the DNA inserted in multicloningsite of pcDNA4 in pcDNA4/V5-His(B).

FIG. 2-1 is graphs showing the results of the luminescence intensitymeasurement in a luciferase split assay in one Example of the presentinvention, which were obtained by using combinations of pFRB-lucC389 topFRB-lucC391 and plucN404-FKBP to plucN417-FKBP. For each sample, theleft bar of the bar graph is the result for the rapamycin-containingculture medium, and the right bar is the result for the DMSO-containingculture medium.

FIG. 2-2 is graphs showing the results of the luminescence intensitymeasurement in a luciferase split assay in one Example of the presentinvention, which were obtained by using combinations of pFRB-lucC392 topFRB-lucC394 and plucN404-FKBP to plucN417-FKBP. For each sample in thebar graph, the left bar is the result for the rapamycin-containingculture medium, and the right bar is the result for the DMSO-containingculture medium.

FIG. 3-1 is graphs showing the results of the luminescence intensitymeasurement in a luciferase split assay in one Example of the presentinvention, which were obtained by using combinations of pFRB-lucC394 topFRB-lucC399 and plucN404-FKBP to plucN417-FKBP. For each sample in thebar graph, the left bar is the result for the rapamycin-containingculture medium, and the right bar is the result for the DMSO-containingculture medium.

FIG. 3-2 is graphs showing the results of the luminescence intensitymeasurement in a luciferase split assay in one Example of the presentinvention, which were obtained by using combinations of pFRB-lucC400 topFRB-lucC403 and plucN404-FKBP to plucN417-FKBP. For each sample in thebar graph, the left bar is the result for the rapamycin-containingculture medium, and the right bar is the result for the DMSO-containingculture medium.

FIG. 3-3 is graphs showing the results of the luminescence intensitymeasurement in a luciferase split assay in one Example of the presentinvention, which were obtained by using combinations of pFRB-lucC404 topFRB-lucC407 and plucN404-FKBP to plucN417-FKBP. For each sample in thebar graph, the left bar is the result for the rapamycin-containingculture medium, and the right bar is the result for the DMSO-containingculture medium.

FIG. 4 is a graph and a table showing the results of the luminescenceintensity measurement in a luciferase split assay in one Example of thepresent invention, which were obtained by using combinations ofpFRB-lucC394 and plucN412-FKBP to plucN416-FKBP. In the table, Rap+ isthe luminescence intensity when binding was induced; DMSO is theluminescence intensity when binding was not induced (namely, thebackground); STDEV-R is the standard deviation when binding was inducedwith Rap+; and STDEV-D is the standard deviation when binding was notinduced with DMSO. For each sample in the graph, the left bar is theresult for the rapamycin-containing culture medium, and the right bar isthe result for the DMSO-containing culture medium.

FIG. 5 is a table showing the results of the luminescence intensitymeasurement in a luciferase split assay in one Example of the presentinvention, which were obtained by using combination of plucN415-FKBP andpFRB-lucC394 and conventional combination of pTlucN-FKBP and pFRB-GlucC.Symbols are as defined above for FIG. 4.

FIG. 6 is a graph showing the results of the comparison of theluminescence intensity, in one Example of the present invention, for thecases when somatostatin was added and not added to HEK293 cells in whichpSSTR2-lucC394 and plucN415-arrestin had been introduced to transientlyexpress SSTR2-lucC394 and lucN415-arrestin, respectively. x axis showspresence (+) and absence (−) of the somatostatin, and y axis showsnumber of photons (×10⁴).

FIG. 7 is a graph showing dose-response curves in one Example of thepresent invention, when HEK293-ARRB2-SSTR2 cell line was stimulated withsomatostatin or its analogs (RIM23052 or BIM23056) at variousconcentrations. x axis shows concentration of each reagent (log [molarconcentration]), and y axis shows number of photons (×10⁴).

FIG. 8 is a graph showing a time-response curve in one Example of thepresent invention, when HEK293-ARRB2-SSTR2 cell line was stimulated with1×10⁻⁶ M of somatostatin, and luminescence was measured with time. xaxis shows time (min), and y axis shows number of photons (×10⁴).

FIG. 9 is a table showing names of the GPCRs used, PCR templates andprimer sequences used in preparing the expression vectors for expressingfusion proteins, ligands used for the stimulation, ligand concentrations(EC50) at which the luminescence was detected (unit: molarconcentration), and the times (T) of the maximum luminescenceobservation after the stimulation, in the experiment conducted for theGPCRs.

DESCRIPTION OF EMBODIMENTS

Next, embodiments of the present invention completed based on thefinding as described above are described in detail by referring toExamples. Unless otherwise noted, methods described in standardprotocols such as J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.),Molecular cloning, a laboratory manual (3rd edition), Cold Spring HarborPress, Cold Spring Harbor, N.Y. (2001); F. M. Ausubel, R. Brent, R. E.Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl (Ed.),Current Protocols in Molecular Biology, John Wiley & Sons Ltd. as wellas their modifications and improvements are used in the embodiments andExamples. When commercially available reagent, kit and assay apparatusare used, protocols attached thereto are used unless otherwise noted.

The objects, features, advantages, and ideas of the present inventionare clear for those skilled in the art from the description of theinvention, and those skilled in the art will be readily capable ofreproducing the invention. The embodiments and Examples as describedbelow are preferable embodiments of the present invention, which arepresented for the purpose of illustration and explanation, and thepresent invention is not limited by these embodiments and Examples. Itis clear for those skilled in the art that the description of thepresent invention can be modified in various ways within the scope andintention of the invention herein described.

[Principle]

The present invention provides a luciferase split assay system with ahigh detection sensitivity. In this assay system, an amino acid sequencecomprising amino acid SEQ ID NO: 1 and an amino acid sequence selectedfrom the group consisting of amino acid SEQ ID NOS: 2 to 6 fromPyrearinus termitilluminans luciferase whose sequence is described inFIG. 1A, are used. Next, methods using this assay system are describedin detail. The luciferase split assay is a technique known in the art,and the procedure not described in this specification may be conductedaccording to common knowledge of those skilled in the art.

First, a first protein (referred to as a first fusion protein) havingthe amino acid sequence of amino acid SEQ ID NO: 1 (this peptide moietyis referred to as lucCmax) and a second protein (referred to as a secondfusion protein) having an amino acid sequence (this peptide moiety isreferred to as lucNmax) selected from the group consisting of amino acidSEQ ID NOS: 2 to 6 are synthesized. It should be noted that the firstprotein and the second protein can bind to each other under particularconditions.

While the fusion proteins can be chemically synthesized for use in theassay system, the fusion proteins may be provided by constructingexpression vectors coding for the fusion proteins and expressing thefusion proteins in the assay system, as will be described below. In sucha case, the fusion proteins may be expressed either transiently orpermanently. The former is preferable when the assay system is an invitro system, and the latter is preferable when the assay system is anin vivo system such as a cell. In each fusion protein, the lucCmax orthe lucNmax may be connected to the protein either directly or via alinker. The linker is preferably a peptide moiety with an adequatelength.

When both of the fusion proteins are introduced in the assay system, thefirst protein and the second protein bind to each other, and as aconsequence, the lucCmax and the lucNmax will be located at positionscapable of undergoing an interaction, and the lucCmax and the lucNmaxwill reconstitute the luciferase to recover luciferase activity so thatthe luciferase is capable of emitting light under adequate luminescentconditions. The luciferase activity may be measured, when the assaysystem is a cell, by adding luciferin to the cell culture, and preparinga cell extract to measure the luciferase activity. In this case, theactivity is readily measurable by using a commercially available EmeraldLuc Luciferase Assay Reagent/Lysis Solution (TOYOBO) or the like.

In this assay system, a luminescence intensity that is about 30 times ormore stronger than the conventional assay is realized when the aminoacid sequence of the lucC is amino acid SEQ ID NO: 1 and the amino acidsequence of the lucN is an amino acid sequence selected from the groupconsisting of amino acid SEQ ID NOS: 2 to 6.

[Construction of Expression Vectors]

As described above, the introduction of the fusion proteins into theassay system can be realized by constructing expression vectors codingfor the fusion proteins and expressing the fusion proteins in the assaysystem.

The expression vectors coding for the fusion proteins can be readilyconstructed by constructing vectors containing DNA coding for the aminoacid sequence selected from the group consisting of amino acid SEQ IDNO: 1 to 6 in advance.

For example, such a vector may be constructed to have DNA coding for thelucNmax having the initiation codon ATG inserted downstream of anexpression promoter which can function in the assay system and followedby a multicloning site immediately downstream and a transcriptiontermination signal further downstream. When DNA coding for the intendedprotein is inserted in frame in the multicloning site, expression of thefusion protein of the lucNmax and the intended protein is facilitated.

Another exemplary vector has a form comprising an expression promoterthat can function in the assay system, the initiation codon ATG, DNAcoding for the multicloning site and the lucCmax, and the transcriptiontermination signal in this order. When DNA coding for the intendedprotein is inserted in frame in the multicloning site, expression of thefusion protein of the lucCmax and the intended protein is facilitated.

Furthermore, an expression vector for the fusion protein having lucCmaxor lucNmax can be readily constructed for the purpose of, for example,detecting a protein-protein interaction when a kit containing a vectorhaving DNA coding for the amino acid sequence of the amino acid SEQ IDNO: 1, namely, the lucCmax and a vector having DNA coding for an aminoacid sequence selected from the group consisting of amino acid SEQ IDNOS: 2 to 6, namely, the lucNmax is prepared.

[Use of the Assay System]

Next, exemplary uses of the assay system of the present invention aredescribed.

First of all, a fusion protein having lucCmax can be detected. Forexample, when a first fusion protein that has been made by fusing atarget protein to be detected with lucCmax exists in the assay system, asecond fusion protein that has been made by fusing a binding proteinthat binds to the target protein with lucNmax is prepared as a probe andis introduced in the assay system. Then, the binding protein in thesecond fusion protein should bind to the target protein in the firstfusion protein; thereby the lucCmax and the lucNmax interact and gainluciferase activity. By detecting the luciferase activity, the targetfusion protein having the lucCmax can be detected. Specifically, anexpression vector expressing the first fusion protein is prepared andintroduced in a cell. Next, an expression vector expressing the secondfusion protein is prepared and introduced in the cell expressing thefirst fusion protein. Then the fusion protein having the lucCmax isdetected by measuring the luciferase activity as described above.

Next, a fusion protein having lucNmax can be detected. For example, if afirst fusion protein that has been made by fusing the target protein tobe detected with lucNmax exists in the assay system, a second fusionprotein that has been made by fusing a binding peptide that binds to thetarget protein with lucCmax is prepared as a probe and is introduced inthe assay system. Then, the binding peptide in the second fusion proteinwill bind to the target peptide in the first fusion protein; thereby thelucNmax and the lucCmax interact and gain luciferase activity. Bydetecting the luciferase activity, the target fusion protein having thelucNmax can be detected. Specifically, an expression vector expressingthe first fusion protein is prepared and introduced in a cell. Next, anexpression vector expressing the second fusion protein is prepared, andintroduced in the cell expressing the first fusion protein. Then thefusion protein having the lucNmax is detected by measuring theluciferase activity as described above.

Further, a complex of a fusion protein having the lucNmax and a fusionprotein having the lucCmax can be detected. When these fusion proteinsform a complex, the lucNmax and the lucCmax interact and gain luciferaseactivity. By detecting the luciferase activity, the complex can bedetected. For detection, the assay system including the complex may beplaced under the conditions wherein the luciferase activity can bedetected. When the assay system is a cell, the luciferase activity maybe measured by the procedure as described above.

Further, binding of the first and second proteins that have mutualbinding ability can be detected, because a first fusion protein and asecond fusion protein are synthesized by fusing a first protein and asecond protein with the lucNmax and the lucCmax in advance respectivelyso that the luciferase activity will be detectable if the first fusionprotein binds to the second fusion protein. It can be examined whetherthe first protein can bind to the second protein by using this method;when the first fusion protein prepared by fusing the lucNmax with thefirst protein and the second fusion protein prepared by fusing thelucCmax with the second peptide are introduced in the assay system, theluciferase activity will be detected if the first protein binds with thesecond protein and the luciferase activity will not be detected if thefirst protein does not bind with the second protein. Specifically,expression vectors expressing the first fusion protein or the secondfusion protein are separately prepared and both of them are introducedin the same cell, and then, if luminescence from the luciferasereconstituted in the cell is observed by measuring the luciferaseactivity as described above, the first protein and the second proteincan be judged to be bound each other, and the first protein and thesecond protein can be judged not to be bound if no luminescence isdetected.

Further, it is possible to screen a protein library for a bindingprotein that is capable of binding to a first protein. Morespecifically, a first fusion protein is prepared by fusing a firstprotein with lucNmax or lucCmax, and second fusion proteins are preparedby fusing second proteins in the protein library with lucCmax orlucNmax, respectively; and when the first fusion protein and the secondfusion proteins are allowed to interact with each other, only the secondfusion proteins having the binding proteins capable of binding with thefirst protein form complexes with the first fusion protein. Accordingly,the second proteins that bind to the first protein can be identified bydetecting luminescence emitted from the complexes. Specifically, a celltransformed with an expression vector which expresses a first fusionprotein comprising a first protein fused with lucNmax is prepared, and acDNA library which has been constructed to express proteins in the formfused with lucCmax is introduced in the cell; then, luciferin is addedto the culture medium, and luminescent cells are identified and cloned.DNAs derived from the library are recovered from the clones, and thegenes expressed are identified to thereby obtain cDNAs of the secondproteins that form the complexes with the first protein.

EXAMPLES Example 1

In this Example, interacting proteins, FKBP (NM_(—)054014) and FRB(NM_(—)019906), which are bound each other in the presence of rapamycin,were fused with lucNs, peptides having the N terminal sequence ofPyrearinus termitilluminans luciferasem and lucCs, peptides having the Cterminal sequence of Pyrearinus termitilluminans luciferase,respectively. It will be shown that the luminescence activity of thecomplex of the interacting proteins varies according to the combinationof lucN and lucC, and is remarkably enhanced when lucNmax (SEQ ID NO: 2to 6; the amino acid sequence of 1st-412nd to 416th amino acid residues)is used in combination with lucCmax (SEQ ID NO: 1; the amino acidsequence of 394th-542nd amino acid residues).

First, PCR was conducted using a cDNA of Pyrearinus termitilluminansluciferase, whose sequence is shown in FIG. 1A, as template and usingthe primers of FIG. 1B to obtain 14 kinds of DNA fragments coding for 14kinds of peptide each having an amino acid sequence from N terminalamino acid residue to the 404th to 417th amino acid residues, which wereobtained by using the pair of N-PtGR-F001 and N-PtGR-R404 to R417, and25 kinds of DNA fragments coding for amino acid sequences from Cterminal amino acid residue to the 389th to 413rd amino acid residues,which were obtained by using C-PtGR-R542 and C-PtGR-F389 to F413. TheDNA coding for the N terminal region was cleaved with HindIII and BamHI,pFKBP was cleaved with BamHI and XhoI, and pcDNA3.1/myc-His (B) wascleaved with HindIII and XhoI to conduct three molecule ligation andthus 14 kinds of plucN-FKBP were prepared. In the meanwhile, the DNAcoding for the C terminal region was cleaved with XhoI and SacII, pFRBwas cleaved with BamHI and XhoI, and pcDNA4/V5-His (B) was cleaved withBamHI and SacII to conduct three molecule ligation and thus 25 kinds ofpFRB-lucC were prepared. pcDNA3.1/myc-His (B) and pcDNA4/V5-His (B) areplasmid vectors having the sequence of SEQUENCE ID NOS: 7 and 8 insertedtherein, respectively. The nucleotide sequence of the DNA inserted inthe multicloning site of pcDNA3.1 is shown in FIG. 1C and the nucleotidesequence of the DNA inserted in the multicloning site of pcDNA4 is shownin FIG. 1D.

For the 339 cases where the luciferase was reconstructed as the aminoacid sequence of the original luciferase (overlapping of the amino acid:0), or with partial overlapping (overlapping of the amino acid: 1 ormore) in the combinations of 14 kinds of plucN-FKBP and 25 kinds ofpFRB-lucC, each pair of the expression vectors was transfected to COSTcells on a 96 well plastic culture dish using TtansIT TransfectionReagents (TAKARA). After about 16 hours from the transfection, theculture medium was replaced with the medium containing 1 μm ofrapamycin; after 24 hours of incubation, ELA (TOYOBO) was added and theluminescence was measured by TriStar LB941 (Berthold Technologies).

As shown in FIGS. 2 and 3, high levels of signal were obtained forpFRB-lucC394, and the signal was the highest in the case ofplucN412-FKBP to plucN416-FKBP. It should be noted that almost no signalwas obtained for pFRB-lucC408 to pFRB-lucC413, and these cases are notshown in the drawings. Further, in FIG. 3, no signal was obtained in thecase of plucN415-FKBP due to the experimental failure.

Accordingly, the experiment was conducted again for plucN412-FKBP toplucN416-FKBP, and as shown in FIG. 4, the highest signals were obtainedfor plucN412-FKBP to plucN416-FKBP at almost the same level.

The luminescence intensity obtained by using the combination ofplucN415-FKBP and pFRB-lucC394 which was shown to be the most suitablewas compared with the luminescence intensity obtained by the combinationof pTlucN-FKBP and pFRB-GlucC which had been accepted as the mostsuitable combination. pTlucN-FKBP is a vector constructed by amplifyingan N terminal fragment of the cDNA of red-emitting Photinus Pyralisluciferase by PCR using the primers as shown below and constructing thevector in the same manner as plucN-FKBP, and pFRB-GlucC is a vectorconstructed by amplifying a C terminal fragment of the cDNA ofgreen-emitting Photinus Pyralis luciferase by PCR using the primers asshown below and constructing the vector in the same manner as pFRB-lucC.

(TlucN-1) (SEQ ID No. 9) 5′AAGCTTGCCATGGTAAAGCGTGAGAAAAATGTC 3′(TlucN-2) (SEQ ID No. 10) 5′GGATCCTCCGCCTCCTCCGCCGTCGTCGATGGCCTC 3′(GlucC-1) (SEQ ID No. 11)5′aggCTCGAGTGGAGGCGGCGGAGGCTGGCTGCACTCTGGCGACTTC 3′ (GlucC-2) (SEQ IDNo. 12) 5′cgcGGGCCCAGCTTAGAAGCCTTCTCCATCAGGGC 3′

As shown in FIG. 5, the luminescence intensity obtained by thecombination of plucN415-FKBP and pFRB-lucC394 was about 30 fold higherthan the conventional combination of plucN415-FKBP and pFRB-lucC394.Thus, the luminescence intensity about 30 fold higher than theconventional luminescence intensity was realized by using the Pyrearinustermitilluminans luciferase and conducting the luciferase split assay byusing the C terminal fragment lucC394 and the N terminal fragmentslucN412 to lucN416.

Example 2

In this Example, somatostatin receptor (SSTR2; somatostatin type 2receptor) (NM_(—)000794) which is a GPCR (G-protein coupled receptor)and β-arrestin (arrestin, beta 2) (NM_(—)004313) were used instead ofthe FKBP and FRB. SSTR2 is a membrane protein on the cell membrane, andwhen somatostatin binds to extracellular domain of the GPCR, theintracellular domain of the SSTR2 binds to β-arrestin that is an adaptermolecule in the cytoplasm, and a signal is transduced downstream.Accordingly, the C terminal of the SSTR2 was bonded to the C terminal ofthe Eluc, and the N terminal of the β-arrestin was bonded to N terminalof the Eluc, and the fusion proteins were expressed in the culturedcells, and somatostatin was added to the cultured cells to examineluminescence from the cells.

First, PCR was conducted by using a human brain cDNA library (TAKARA) astemplate with the primers as shown below to obtain DNA fragments codingfor arrestin and the SSTR2.

ARRB2-nestF2: (SEQ ID NO: 54) AAAGGATCCATGGGGGAGAAACCCGGGACCAGGGTCTARRB2-nestR-Eco: (SEQ ID NO: 55) AAGAATTCCAGCAGAGTTGATCATCATAGTSSTR2_start_Bam: (SEQ ID NO: 56) TTGGATCCATGGACATGGCGGATGAGCCACSSTR2_R1107end_XhoI: (SEQ ID NO: 57)TTTCTCGAGCCGATACTGGTTTGGAGGTCTCCATTG

The DNA coding for the arrestin was cleaved with BamHI and EcoRI, andligated to the plucN415 that had been cleaved with BamHI and EcoRI andplucN415-arrestin was obtained. The plucN415 used had been obtained bycleaving the plucN415-FKBP in Example 1 with HindIII and BamHI andligating with pcDNA3.1/myc-His(B) cleaved with HindIII and BamHI.

In the meanwhile, the DNA fragment coding for the SSTR2 was cleaved withBamHI and XhoI, inserted in the multicloning site of pcDNA4/V5-His(B),and pSSTR2 was obtained. Then, the DNA coding for lucC394 with thelinker whose length was extended to 22 amino acids was cleaved with XhoIand SacII, inserted at XhoI-SacII site of the pSSTR2 and pSSTR2-lucC394was obtained. It is to be noted that the linker length of the lucC394was extended to 22 amino acids step by step by conducting PCR usingpFRB-lucC394 as template and linker C12-F-XhoI (SEQ ID NO: 58) andPtGR-R542-SacII (SEQ ID NO: 61) as primers, cleaving the PCR productwith XhoI and SacII, and inserting the fragment at the XhoI-SacII siteof the pSSTR2; conducting PCR using this producer as template and linkerC17-F-XhoI (SEQ ID NO: 59) and PtGR-R542-SacII (SEQ ID NO: 61) asprimers, cleaving the PCR product with XhoI and SacII, and inserting thefragment at XhoI-SacII site of the pSSTR2; and finally, conducting PCRusing this product as template and linker C22-F-XhoI (SEQ ID NO: 60) andPtGR-R542-SacII (SEQ ID NO: 61) as primers, cleaving the PCR productwith XhoI and SacII, and inserting the fragment at XhoI-SacII site ofthe pSSTR2.

linkerC12-F-XhoI: (SEQ ID NO: 58)AGGCTCGAGTGGCGGTGGAGGTAGTGGAGGCGGCGGAACAAA linkerC17-F-XhoI: (SEQ ID NO:59) AGGCTCGAGTGGTGGTGGGGGCAGTGGCGGTGGAGGTAGTGG linkerC22-F-XhoI: (SEQ IDNo. 60) AGGCTCGAGTGGAGGTGGCGGTTCTGGTGGTGGGGGCAGTGGCGGT PtGR-R542-SacII:(SEQ ID No. 61) TTTCCGCGGCAGCTTAGAAGCCTTCTC

The pSSTR2-lucC394 and plucN415-arrestin thus prepared were transfectedto the HEK293 cells cultured in a 96 well plastic culture dishes byusing TtansIT Transfection Reagents (TAKARA). After about 40 hours fromthe transfection, the cells were incubated in a culture mediumcontaining 1 μm of somatostatin for 12 minutes, ELA (TOYOBO) was added,and the luminescence was measured with TriStar LB941 (BertholdTechnologies). The luminescence was also measured for the control cellwith no addition of the somatostatin, and the results were compared. Asshown in FIG. 6, addition of the somatostatin resulted in thesignificant enhancement of the luminescence, whose intensity was eighttimes.

Next, HEK293 cells which had been transfected with plucN415-arrestinusing 6 cm plastic culture dishes as described above were cultured for20 days in a culture medium containing 0.8 mg/mL of G418 and an HEK293cell line (HEK293-ARRB2) capable of constantly expressinglucN415-arrestin was prepared. This cell line was transfected withpSSTR2-lucC394 as described above, and the cells were cultured for 20days in a culture medium containing 0.8 mg/mL of G418 and 0.04 mg/mL ofZeocin and an HEK293 cell line (HEK293-ARRB2-SSTR2) capable ofconstantly expressing lucN415-arrestin and SSTR2-lucC394 was prepared.

The cells were cultured in a 96 well plastic culture dish, and afterstimulating the cells for 12 minutes with somatostatin or its analog(RIM23052 or BIM23056) at various concentrations, luminescence wasmeasured as described above. A dose-response curve showing relationshipof the luminescence intensity and the ligand concentration was made fromthe results obtained.

As shown in FIG. 7, in the case of somatostatin, enhancement in theluminescence was observed at a concentration of 3×10⁻⁹ to 3×10⁻⁷ M, andthe enhancement was not enhanced at the higher somatostatinconcentration. In the case of the somatostatin analogues, suchenhancement in the luminescence was not observed at the sameconcentration. Thus, quantitative assay of the ligand activity to thereceptor was enabled using the assay system in which the luciferasesplit assay of the present invention is applied to the receptor and theintracellular binding element.

When HEK293-ARRB2-SSTR2 was stimulated with 1×10⁻⁶ M somatostatin andthe luminescence was measured at 3 minutes, 6 minutes, 12 minutes, 15minutes, 30 minutes, 40 minutes, 50 minutes, and 90 minutes after thestimulation, the luminescence reached 90% of the maximum luminescence in5 minutes and the luminescence reached its maximum in 12 minutes, asshown in FIG. 8. After 12 minutes, the luminescence gradually reduced.However, the level of 80% of the maximum luminescence was stillmaintained after 90 minutes. Thus, the assay system employing theluciferase split assay of the present invention has enabled more promptdetection compared to the conventional protein-protein interactiondetection system.

The luciferase split assay of the present invention can be applied tothe GPCR other than SSTR2, namely ADRB2 (adrenergic beta 2 receptor,surface) (NM_(—)000024), AGTRL1 (apelin receptor) (NM_(—)00516), EDNRB(endothelin receptor type B) (NM_(—)000115), and CCKBR (cholecystokininB receptor) (NM_(—)17685), and the results shown in FIG. 9 were obtainedin similar experimental systems.

INDUSTRIAL APPLICABILITY

The present invention has enabled to provide a split luciferase assaysystem having a remarkably high detection sensitivity.

What is claimed is:
 1. A fusion protein consisting of the amino acidsequence of SEQ ID NO: 1 and a peptide not derived from Pyrearinustermitilluminans luciferase.
 2. A fusion protein consisting of an aminoacid sequence selected from the group consisting of SEQ ID NOS: 2 to 6fused with a peptide not derived from Pyrearinus termitilluminansluciferase.
 3. A complex of a fusion protein of claim 1 and a fusionprotein of claim
 2. 4. The complex of claim 3 wherein said complexcomprises a first fusion protein, said first fusion protein comprising afirst protein fused to the amino acid of SEQ ID NO: 1; and a secondfusion protein, said second fusion protein comprising a second proteinfused to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2 to 6, wherein said first and second proteins interact toform a complex.
 5. The complex of claim 4 wherein said first and secondproteins have mutual binding.
 6. The complex of claim 4 wherein saidcomplex further comprises a moiety that binds the first and secondproteins.
 7. The complex of claim 4 wherein either of said first proteinand second protein is FK506-binding protein (FKBP) or is FKBP-bindingdomain (FRB).
 8. The complex of claim 7 wherein the first protein isFKBP, and the second protein is FRB.
 9. The complex of claim 6 whereinthe moiety is rapamycin.
 10. The complex of claim 4 wherein either ofsaid first protein and second protein is a somatostatin receptor or isbeta-arrestin.
 11. The complex of claim 6 wherein the moiety is aG-protein coupled receptor.
 12. The complex of claim 11 wherein thefirst protein is somatostatin type 2 receptor, the second protein isbeta-arrestin, and the C terminus of the somatostain type 2 receptor islinked to SEQ ID NO: 1 and the N terminus of the beta-arrestin is linkedto an amino acid sequence selected from the group consisting if SEQ IDNOs: 2 to
 6. 13. A kit for detecting the binding of a first fusionprotein and a second fusion protein in the presence of a moiety thatbinds to both the first and second fusion proteins, said kit comprisinga first fusion protein, said first fusion protein comprising a firstprotein fused to the amino acid sequence of SEQ ID NO: 1; and a secondfusion protein, said second fusion protein comprising a second proteinfused to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2 to 6, wherein said first and second proteins interact withsaid moiety to form a ternary complex.